Fluorescent labeling, one of the most dominant non-radioactive biological detection modalities, is a highly desirable method for the detection of nucleic acids. For example, this method is useful in automated DNA sequencing, in situ detection of hybridization, detection of PCR products, structural studies, and any of several other applications (G. C. Howard, Methods in Nonradioactive Detection, 1993; L. J. Kricka, Nonisotopic Probing, Blotting and Sequencing, 1995). In the past, the labelling of oligonucleotides has been most conveniently accomplished on an automated synthesizer by introduction of a derivatized deoxyuridine phosphoramidite or a linker phosphoramidite bearing a protected primary amine or thiol group. Consequently, the preparation of a labelled oligonucleotide requires synthesis of an oligonucleotide bearing the aforementioned modifications. The oligomer is deprotected, liberating the nucleophile, which can react with a fluorescent label or other tags. This procedure entails at least a partial purification of the deprotected oligomer, reaction with the fluorescent dye derivative (such as a fluorescent dye succinimidyl ester, isothiocynate, sulfonyl chloride or maleimide, etc.), removal of the excess reagent, and purification of the labelled oligomer. The addition of the label (e.g., a fluorescent dye) by an extra step, and the additional step for purifying the labeled product are often tedious, which increases synthesis time considerably (S. Ochet, et al., Tetrahedron 1988, 43, 3481; J. Haralambidis, et al., Tetrahedron Lett. 1987, 28, 5199; M. S., Urdea et al., Nucl. Acids Res. 1988, 16, 4937; S. Le Brun, et al., J. Biochemie 1989, 71, 319). The overall process requires approximately two days, compared to a few hours for preparation of the corresponding unlabeled oligonucleotides. In contrast, fluorescent dye phosphramidites enable the rapid preparation of fluorescent oligonucleotides just as the unlabeled oligonucleotides.
Fluoresceins have long been known for their fluorescent properties and are quite useful in labelling biomolecules. They have a very high absorbance with excellent fluorescence quantum yields, considering to be among the most popular fluorescent dyes used in labelling oligonucleotides. One prior art method described a method to link a fluorescein to oligonucleotides through fluorescein phosphoramidite chemistry (U.S. Pat. No. 5,583,236). However, all the oligonucleotides prepared from the known fluorescein phosphoramidites have significantly reduced fluorescence at physiological pH (˜7.0) due to the high pKa of fluoresceins used for preparing fluorescein phosphoramidites. Another drawback for the existing fluorescein-labeled oligonucleotides results from the low photostability of the fluoresceins used for preparing fluorescein phosphoramidites, which limits the assay performance with the fluorescein-labeled oligonucleotides. The third drawback for the existing fluorescein-labeled oligonucleotides is that their short excitation and emission wavelengths severely overlap with the wavelengths of many natural substances in cells or biological samples, resulting in high assay background.
The compound of this invention eliminates the above described drawbacks of the existing fluorescein phosphoramidites. The oligonucleotides prepared from these new fluorescein derivatives have red-shifted wavelengths, reduced pKa and enhanced photostability. Under the same conditions, the fluorescence detections performed with the oligonucleotides prepared from the new fluorescein derivatives of this invention demonstrate low assay background, high sensitivity and enhanced photostability.